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Related Experiment Videos

Solid KHT tumor dispersal for flow cytometric cell kinetic analysis

M G Pallavicini, L J Folstad, C Dunbar

    Cytometry
    |September 1, 1981
    PubMed
    Summary

    Bacterial neutral protease effectively disperses KHT tumors into single cells for flow cytometry and cell survival studies. This method yields more cells and higher plating efficiency than traditional trypsin/DNase procedures.

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    Area of Science:

    • Biotechnology
    • Cancer Research
    • Cell Biology

    Background:

    • Accurate cell kinetic analysis and clonogenic cell survival assays are crucial for understanding tumor behavior and treatment response.
    • Traditional tumor dispersal methods, such as trypsin/DNase, can be inefficient, yielding suboptimal cell numbers and potentially affecting cell viability.
    • Developing improved methods for generating single-cell suspensions from solid tumors is essential for advancing cancer research.

    Purpose of the Study:

    • To evaluate the efficacy of bacterial neutral protease for dispersing KHT solid tumors into single-cell suspensions.
    • To compare neutral protease with a standard trypsin/DNase procedure regarding cell yield, clonogenic survival, and cell kinetic analysis.
    • To assess the suitability of neutral protease for flow cytometry and cell survival measurements, including drug-perturbed studies.

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    Main Methods:

    • KHT solid tumors were dispersed using either bacterial neutral protease or a trypsin/DNase procedure under standardized conditions.
    • Evaluated parameters included cell yield, clonogenic cell survival in a semisolid agar system, and DNA distribution analysis via flow cytometry.
    • Assessed radioactive precursor incorporation rates (tritiated thymidine) and investigated phase-specific cell loss during the cell cycle.

    Main Results:

    • Neutral protease yielded approximately four times more single cells compared to the trypsin/DNase method.
    • Clonogenic cell survival was approximately 1.5-fold higher with neutral protease dispersal.
    • Both methods yielded comparable results for DNA distribution analysis and cell cycle kinetic parameters, with no observed phase-specific cell loss.

    Conclusions:

    • Bacterial neutral protease is a superior method for dispersing KHT tumors, providing significantly higher cell yields and improved plating efficiency.
    • Neutral protease generates high-quality single-cell suspensions suitable for routine cell kinetic analysis by flow cytometry and clonogenic cell survival assays.
    • The high cell yield achieved with neutral protease is advantageous for techniques like electronic cell sorting, which require large cell numbers.